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ABSTRACT Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The _Escherichia coli_ Cas1–Cas2 complex mediates spacer

acquisition _in vivo_, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to

yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA

with free 3′-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting

AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the

significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity. Access through your institution Buy or subscribe This is a preview

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ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS GENOME EXPANSION BY A CRISPR

TRIMMER-INTEGRASE Article Open access 14 June 2023 HISTONES DIRECT SITE-SPECIFIC CRISPR SPACER ACQUISITION IN MODEL ARCHAEON Article 07 August 2023 ALTERNATIVE FUNCTIONS OF CRISPR–CAS

SYSTEMS IN THE EVOLUTIONARY ARMS RACE Article 06 January 2022 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE64552 DATA DEPOSITS Sequencing data are deposited in Gene

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ACKNOWLEDGEMENTS We are grateful to M. Chung, P. J. Kranzusch and A.V. Wright for technical assistance and members of the Doudna laboratory and J. Cate for discussions. This project was

funded by US National Science Foundation grant no. 1244557 to J.A.D. and by NIH grant AI070042 to A.E. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley,

supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303. J.K.N. is supported by a US National Science Foundation Graduate Research Fellowship and a UC Berkeley Chancellor’s

Graduate Fellowship. A.S.Y.L. is supported as an American Cancer Society Postdoctoral Fellow (PF-14-108-01-RMC). J.A.D. is an Investigator of the Howard Hughes Medical Institute and a member

of the Center for RNA Systems Biology. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California

94720, USA, James K. Nuñez, Amy S. Y. Lee & Jennifer A. Doudna * Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA, Amy S. Y. Lee & 

Jennifer A. Doudna * Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, 02115, Massachusetts, USA Alan

Engelman * Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA, Jennifer A. Doudna * Department of Chemistry, University of California,

Berkeley, Berkeley, California 94720, USA, Jennifer A. Doudna * Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA Jennifer A. Doudna

Authors * James K. Nuñez View author publications You can also search for this author inPubMed Google Scholar * Amy S. Y. Lee View author publications You can also search for this author

inPubMed Google Scholar * Alan Engelman View author publications You can also search for this author inPubMed Google Scholar * Jennifer A. Doudna View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS J.K.N. performed the biochemical experiments. A.S.Y.L. processed and analysed the high-throughput sequencing data. J.K.N., A.S.Y.L.,

A.E. and J.A.D. designed the study, analysed the data and wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Jennifer A. Doudna. ETHICS DECLARATIONS COMPETING INTERESTS J.A.D. and

J.K.N. have filed a related patent application. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 THE INTEGRATION REACTION IS DEPENDENT ON THE PRESENCE OF PROTOSPACERS, LOW SALT AND

DIVALENT METAL IONS. A, _In vitro_ integration assay alongside EcoRI- and Nb.BbvCI nickase-treated pCRISPR. B, Salt-dependence assay using Cas1 or Cas2 only and Cas1+Cas2. The titration

corresponds to 0, 25, 50, 100 and 200 nM KCl, in addition to the salt carried in from the reaction reagents. C, Integration assays in the presence of 10 mM EDTA, Mg2+, Mn2+ or no additive.

D, Integration assays with increasing protospacer concentrations. E, A comparison of post-reaction treatments as indicated. The data presented in A–E are representative of at least three

replicates. EXTENDED DATA FIGURE 2 CAS1 REQUIRES CAS2 FOR ROBUST PROTOSPACER INTEGRATION. A, Schematic of the integration assays using 32P-labelled protospacers (PDB code 4P6I for

Cas1–Cas2). B, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. C, Same as B except in the presence of 10

 mM MgCl2. The data presented in B and C are representative of at least three replicates. EXTENDED DATA FIGURE 3 THE CATALYTIC ACTIVITY OF CAS1 IS REQUIRED FOR INTEGRATION. A, Close-up view

of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I). B, Integration assays of purified Cas1 active site mutants complexed with wild-type Cas2. C, The

same as B except using radiolabelled protospacers. The data presented in B and C are representative of at least three replicates. EXTENDED DATA FIGURE 4 BAND X CORRESPONDS TO TOPOISOMERS OF

PCRISPR. A, Agarose gel of purified relaxed and band X integration products. B, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a

pre-stained agarose gel. C, Same as B except ethidium bromide staining was performed after electrophoresis. D, PCR amplification products of various segments of pCRISPR using the relaxed,

band X or pCRISPR template shown in A. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data

presented in A–D are representative of at least three replicates. EXTENDED DATA FIGURE 5 CAS1 CATALYSES THE DISINTEGRATION OF HALF-SITE INTEGRATED PROTOSPACERS. A, Schematic of the four

strands constituting the Y DNA substrate used in the disintegration assays. B, Native polyacrylamide gel analysis of the annealing products with either strand A or strand C radiolabelled. C,

Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with strand A labelled. D, Denaturing gel analysis of the disintegration assay products with

strand A labelled. EXTENDED DATA FIGURE 6 CAS1–CAS2 CAN INTEGRATE VARIOUS LENGTHS OF DOUBLE-STRANDED DNA WITH BLUNT- OR 3′-OVERHANG ENDS INTO A SUPERCOILED TARGET PLASMID. A, Integration

assays using the indicated lengths of protospacer DNA. B, Integration assays using varying 5′ or 3′ overhang lengths. C, D, A comparison of integration assays using pCRISPR or

Nb.BbvCI-nicked pCRISPR target. E, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the

cyan arrows correspond to the band X product. The data presented in A–E are representative of at least three replicates. EXTENDED DATA FIGURE 7 CAS1 TYROSINE MUTANTS SUPPORT INTEGRATION

ACTIVITY _IN VITRO_. A, A close-up of the Cas1 active site with the tyrosine residues labelled in blue. B, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine

residues mutated to alanine in this study. C, Radiolabelled protospacer integration assay of Cas1 tyrosine mutants complexed with wild-type Cas2. The gel presented in C is representative of

at least three replicates. EXTENDED DATA FIGURE 8 HIGH-THROUGHPUT SEQUENCING OF INTEGRATION PRODUCTS REVEALS SEQUENCE-SPECIFIC INTEGRATION. A, Schematic of the workflow for high-throughput

sequencing analysis of the integration sites. B, Raw map of the total reads along pCRISPR before collapsing into single peaks of protospacer–pCRISPR junctions depicted in Fig. 4. C, Same as

B, except for the pUC19 target. D, Sequence of the leader-end of the CRISPR locus in _E. coli_. E, F, WebLogo analysis from the −5 to +5 positions surrounding the protospacer integration

sites on the plus (E) and minus (F) of pCRISPR. The arrow points to the nucleotide that is covalently joined to the protospacer. G, H, Same as E, F, except for the pUC19 target. EXTENDED

DATA FIGURE 9 CAS1–CAS2 CORRECTLY ORIENTS THE PROTOSPACER DNA DURING INTEGRATION. A–F, Mapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide

ends ‘wild-type’ 3′ C and 3′ T (A), 3′ A and 3′ T (C), and 3′ C and 3′ C (E). The red arrow in C and E points to the nucleotide change in the protospacer DNA compared to the ‘wild-type’

sequence in A. The protospacer DNA 3′ nucleotide and the CRISPR locus strand biases in A, C, E are plotted in B, D and F, respectively, as percentages of integration events within the CRISPR

locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and _P_ < 0.0001 by chi-square test. The _n_ values

for B, D and F are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively. EXTENDED DATA FIGURE 10 MODEL OF THE CRISPR–CAS ADAPTIVE IMMUNITY PATHWAY IN _E. COLI_. Mature

double-stranded protospacers bearing a 3′ C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5′G/3′C as the

first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific

recognition of the 3′-TTC-5′ PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to

the inability for the crRNA to recognize the strand with the 3′-TTC-5′ PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly

orienting the protospacer during integration. POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT

SLIDE FOR FIG. 5 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Nuñez, J., Lee, A., Engelman, A. _et al._ Integrase-mediated spacer acquisition during

CRISPR–Cas adaptive immunity. _Nature_ 519, 193–198 (2015). https://doi.org/10.1038/nature14237 Download citation * Received: 06 November 2014 * Accepted: 15 January 2015 * Published: 18

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